ERF113 Antibody

What is ERF113 and what role does it play in plant biology?

ERF113 (Ethylene-Responsive Factor 113) belongs to the AP2/ERF transcription factor family that regulates various plant biological processes. This transcription factor contains a conserved DNA-binding domain that recognizes GCC box elements in promoters of target genes. ERF113 plays crucial roles in:

• Stress response signaling, particularly drought resistance as demonstrated in soybean studies where GmERF113 positively regulates drought tolerance

• Ethylene-mediated signaling pathways, functioning downstream of EIN3/EIL1 transcription factors

• Potential interaction with plant defense mechanisms, as many ERFs are involved in pathogen response pathways

• Transcriptional activation through interaction with mediator complexes, though specific interactions for ERF113 require further characterization

In soybean, GmERF113 has been shown to enhance drought tolerance through direct activation of GmPR10-1 and affects ABA (abscisic acid) content, demonstrating its importance in stress response mechanisms .

What are the key considerations for ERF113 antibody validation?

Proper validation of ERF113 antibodies requires multiple approaches to ensure specificity and reliability:

• Knockout/knockdown controls: Testing antibodies in ERF113-RNAi plant lines is essential for validating specificity, as demonstrated in transgenic soybean studies

• Overexpression controls: Using ERF113-overexpressing plants provides a positive control with higher protein levels for antibody validation

• Western blot analysis: Confirming proper molecular weight (typically in the range of 20-30 kDa for ERF proteins) and absence of non-specific bands

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of ERF113 to confirm consistent results

• Immunoprecipitation followed by mass spectrometry: Confirming the identity of the immunoprecipitated protein

• Cross-reactivity assessment: Testing antibody specificity against closely related ERF family members, particularly important due to the high conservation within the AP2/ERF domain

Following the approaches used for validating other plant transcription factor antibodies, proper controls should include unstained cells, negative cells (not expressing the protein), isotype controls, and secondary antibody controls .

What sample preparation techniques are optimal for ERF113 detection?

Effective sample preparation is critical for successful ERF113 detection:

For plant tissue extraction:

• Use fresh tissue whenever possible, with rapid freezing in liquid nitrogen to preserve protein integrity

• Include protease inhibitors, phosphatase inhibitors (if studying phosphorylation status), and reducing agents in extraction buffers

• Optimize nuclear extraction protocols as transcription factors are often low-abundance nuclear proteins

• Consider specialized extraction buffers containing 0.1% SDS or non-ionic detergents to aid in membrane-associated protein extraction

For protein preservation:

• Maintain cold temperatures throughout extraction to prevent degradation

• Use denaturing conditions for western blotting applications

• For native conditions (needed for immunoprecipitation or ChIP assays), optimize salt concentration and detergent types

For fixation in immunocytochemistry:

• Cross-linking with 1.5 mM EGS followed by formaldehyde fixation (similar to protocols used for other nuclear proteins)

• Careful permeabilization with 0.1% Triton X-100 or similar detergents to allow antibody access to nuclear proteins

How can ChIP assays with ERF113 antibodies be optimized for plant tissues?

Chromatin immunoprecipitation (ChIP) is a powerful technique for identifying direct target genes of ERF113. For optimal results with plant tissues:

• Chromatin preparation and fixation:

  • Follow established Dual-X-ChIP protocols with sequential crosslinking using EGS (1.5 mM, 30 minutes) followed by formaldehyde (10 minutes)

  • Use appropriate amounts of chromatin (25-50 μg per immunoprecipitation)

  • Ensure thorough tissue disruption by grinding in liquid nitrogen

• Antibody selection and amount:

  • Use 5-10 μg of purified ERF113 antibody per ChIP reaction

  • Include appropriate controls (normal IgG from the same species as the primary antibody)

• Immunoprecipitation conditions:

  • Use 25-50 μl of Protein A/G Dynabeads for each IP reaction

  • Perform adequate washing steps to reduce background

  • Process controls identically to experimental samples

• Analysis methods:

  • Quantify immunoprecipitated DNA by real-time PCR using primers targeting putative GCC-box containing promoters

  • Design primers for regions 500-2000 bp upstream of transcription start sites

  • Use appropriate normalization methods (percent input or fold enrichment over IgG)

• Troubleshooting high background:

  • Increase washing stringency with higher salt concentrations

  • Pre-clear chromatin with beads before adding antibody

  • Block beads with BSA to prevent non-specific binding

This approach has been demonstrated effective for identifying direct target genes of ERF transcription factors in plants, as seen in studies of the GmERF113-GmPR10-1 pathway .

What strategies should be employed for detecting post-translational modifications of ERF113?

Post-translational modifications (PTMs) of ERF113 may regulate its activity, subcellular localization, and stability. Effective detection strategies include:

• Phosphorylation analysis:

  • Use phospho-specific antibodies targeting predicted phosphorylation sites

  • Apply strategies similar to those used for ERK1 phospho-specific antibodies, where epitope selection focuses on specific phosphorylated residues

  • Confirm phosphorylation status with λ-phosphatase treatment controls

  • Consider targeted mass spectrometry approaches to identify phosphorylation sites

• Ubiquitination detection:

  • Co-immunoprecipitation with ERF113 antibodies followed by ubiquitin detection

  • Use proteasome inhibitors (MG132) to stabilize ubiquitinated forms, as demonstrated in EBF1/EBF2 studies

• Protein stability assessment:

  • Cycloheximide chase assays using ERF113 antibodies to monitor protein turnover

  • Compare protein stability under different stress conditions

  • Investigate potential regulation by E3 ligases similar to how SDIR1 affects EBF1/EBF2 stability in ethylene signaling

• Subcellular localization changes:

  • Track ERF113 movement between cytosol and nucleus using immunofluorescence

  • Fractionate cell compartments biochemically and analyze with western blotting

These approaches enable detailed investigation of regulatory mechanisms controlling ERF113 function beyond transcriptional regulation.

How can ERF113 protein-protein interactions be investigated using antibody-based approaches?

Understanding ERF113 protein interactions is crucial for elucidating its function in transcriptional complexes. Effective methods include:

• Co-immunoprecipitation (Co-IP):

  • Use purified ERF113 antibodies at 1/20 dilution for immunoprecipitation

  • Process 10-20 μg of plant tissue lysate for each IP reaction

  • For interacting proteins detection, use western blotting with specific antibodies

  • Include appropriate controls (IgG from same species as primary antibody)

  • Consider using VeriBlot for IP secondary antibodies to reduce heavy/light chain interference

• Proximity ligation assays (PLA):

  • Use combinations of ERF113 antibodies with antibodies against suspected interacting proteins

  • Visualize interactions with fluorescence microscopy

  • Quantify interaction signals to assess interaction strength

• BiFC confirmation:

  • After identifying potential interactors through Co-IP, confirm direct interactions using bimolecular fluorescence complementation

  • This provides spatial information about where in the cell interactions occur

• Chromatin-associated protein complexes:

  • Use sequential ChIP (Re-ChIP) to identify co-binding transcription factors

  • Combine with proteomics approaches to identify complete transcriptional complexes

When investigating ERF113 interactions, focus on potential partners in transcriptional complexes such as mediator proteins (like MED25), which are known to interact with ERF family members through the EDLL motif .

What are the optimal western blotting conditions for detecting ERF113?

Western blotting for low-abundance transcription factors like ERF113 requires optimization:

Sample preparation:

• Use nuclear extraction protocols to enrich for transcription factors

• Load adequate protein amounts (typically 20-50 μg of total protein)

• Include phosphatase inhibitors if studying phosphorylation status

Electrophoresis and transfer:

• Use 10-12% SDS-PAGE gels for optimal separation

• Transfer at lower voltage (30V) overnight at 4°C for efficient transfer of transcription factors

• Consider PVDF membranes with 0.2 μm pore size for better protein retention

Antibody conditions:

• Block membranes with 5% non-fat dry milk (NFDM) in TBST

• Use purified ERF113 antibody at approximately 1/5000 dilution (optimize based on antibody quality)

• Incubate primary antibody overnight at 4°C

• Use HRP-conjugated secondary antibodies at 1/1000-1/5000 dilution

Detection and troubleshooting:

• Use enhanced chemiluminescence with longer exposure times if signal is weak

• If multiple bands appear, validate specificity using knockout/RNAi plant lines

• For weak signals, consider signal amplification systems or more sensitive detection methods

The predicted molecular weight of ERF113 should be confirmed based on amino acid sequence, typically in the range of 20-30 kDa for most ERF proteins.

How should immunohistochemistry protocols be optimized for ERF113 detection in plant tissues?

Effective immunohistochemistry for nuclear transcription factors in plant tissues requires specific considerations:

• Tissue fixation and processing:

  • Fix tissues with 4% paraformaldehyde

  • For paraffin-embedded sections, use heat-mediated antigen retrieval with Tris/EDTA buffer pH 9

  • For frozen sections, a concentration range of 1.0-10.0 μg/mL antibody is typically optimal

• Antibody incubation:

  • Use purified primary antibody at 1/1000 dilution (optimize as needed)

  • Incubate sections with primary antibody overnight at 4°C

  • Use HRP-conjugated secondary antibodies at 1/500 dilution

• Signal detection:

  • For chromogenic detection, use DAB and counterstain with hematoxylin

  • For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies

• Controls:

  • Include negative controls using tissue known not to express ERF113

  • Use isotype control antibodies at the same concentration as primary antibody

  • Include a peptide competition assay to confirm specificity

• Troubleshooting:

  • For high background, increase blocking time and washing steps

  • For weak signal, optimize antigen retrieval methods

  • For non-specific binding, increase antibody dilution or use more stringent washing

Following these guidelines will help achieve specific nuclear staining of ERF113 in plant tissues.

What approaches can be used to measure ERF113 protein levels during stress responses?

Quantifying ERF113 protein levels during stress responses requires sensitive and reproducible methods:

• Western blot quantification:

  • Use internal loading controls (housekeeping proteins like actin or GAPDH)

  • Apply time-course sampling to capture dynamic changes

  • Normalize ERF113 band intensity to loading controls using image analysis software

  • Include standard curves with recombinant protein if absolute quantification is needed

• Flow cytometry approach:

  • Fix cells with 80% methanol for intracellular protein detection

  • Permeabilize with suitable detergents

  • Use fluorophore-conjugated secondary antibodies (e.g., FITC-conjugated anti-rabbit IgG)

  • Include isotype controls to set proper gating strategies

• ELISA-based quantification:

  • Develop sandwich ELISA using two antibodies recognizing different epitopes

  • Create standard curves with recombinant ERF113 protein

  • Process samples consistently to minimize variation

• Imaging-based quantification:

  • Use immunofluorescence with consistent imaging parameters

  • Quantify nuclear fluorescence intensity as measure of protein levels

  • Include reference standards in each experiment

These methods allow researchers to quantitatively assess how ERF113 protein levels change in response to environmental stresses, complementing transcript-level analyses.

How can research studies differentiate between closely related ERF family members?

Distinguishing between ERF113 and closely related ERF family members requires careful experimental design:

• Antibody epitope selection:

  • Choose epitopes outside the conserved AP2/ERF domain

  • Target regions with maximum sequence divergence among family members

  • Consider using antibodies against synthetic peptides unique to ERF113

• Validation experiments:

  • Perform western blots on recombinant proteins of multiple ERF family members

  • Test for cross-reactivity against the most closely related ERFs

  • Use ERF113-specific knockdown/knockout lines to confirm specificity

• Combined approaches:

  • Correlate protein detection with transcript levels using RT-qPCR

  • Confirm identity of detected proteins through immunoprecipitation followed by mass spectrometry

  • Use bioinformatics to predict potential cross-reactivity based on epitope conservation

• Specific analytical techniques:

  • 2D gel electrophoresis to separate proteins based on both molecular weight and isoelectric point

  • Compare detection patterns between different antibodies targeting distinct epitopes

  • Use competitive binding assays with specific peptides

These approaches help ensure that research findings are specifically attributed to ERF113 rather than related family members.

How can ERF113 antibodies facilitate identification of novel target genes?

ERF113 antibodies can be powerful tools for discovering previously unknown target genes through several approaches:

• ChIP-sequencing:

  • Perform chromatin immunoprecipitation with ERF113 antibodies followed by next-generation sequencing

  • Analyze enriched sequences for presence of GCC-box or related motifs

  • Apply peak calling algorithms to identify binding regions

  • Compare binding profiles under different stress conditions

• CUT&RUN or CUT&Tag approaches:

  • These newer methods offer improved signal-to-noise ratio compared to traditional ChIP

  • Require less starting material and can provide higher resolution

  • Use the same validated ERF113 antibodies as used in ChIP

• Validation of direct targets:

  • Confirm binding through ChIP-qPCR on candidate promoters

  • Use dual-luciferase reporter assays to validate functional significance of binding

  • Similar to approaches used to demonstrate GmERF113 directly activates GmPR10-1

• Integration with transcriptomics:

  • Combine ChIP-seq data with RNA-seq from ERF113 overexpression and knockout/knockdown lines

  • Identify genes that are both bound by ERF113 and differentially expressed

  • Use this integrated approach to build gene regulatory networks

This methodological framework has been successfully employed to identify direct targets of other ERF family members and can be adapted specifically for ERF113 .

What experimental design considerations are important for studying ERF113 in different plant species?

When investigating ERF113 across different plant species, researchers should consider:

• Sequence conservation assessment:

  • Perform sequence alignment of ERF113 homologs across target species

  • Identify regions of high conservation that may be recognized by the same antibody

  • Consider generating species-specific antibodies if conservation is low

• Cross-reactivity testing:

  • Validate antibody binding using western blots on protein extracts from different species

  • Optimize antibody concentration for each species independently

  • Perform peptide competition assays to confirm specificity in each species

• Control selection:

  • Include appropriate positive controls (overexpression lines) and negative controls (knockdown lines) for each species

  • Consider using heterologous expression systems to test antibody reactivity

• Protocol optimization:

  • Modify extraction and immunoprecipitation buffers based on species-specific characteristics

  • Adjust fixation and permeabilization conditions for immunohistochemistry

  • Validate CHIP protocols independently for each species

• Functional conservation studies:

  • Compare binding profiles and target genes across species

  • Assess whether ERF113 functions are conserved using complementation studies

  • Determine if post-translational modifications are similarly regulated

These considerations are essential for comparative studies of ERF113 function across plant lineages.

How can ERF113 antibodies be used to investigate protein stability and turnover?

Understanding ERF113 protein stability and regulatory mechanisms requires specific experimental approaches:

• Cycloheximide chase assays:

  • Treat plant tissues with cycloheximide to inhibit new protein synthesis

  • Collect samples at various time points and analyze by western blotting with ERF113 antibodies

  • Calculate protein half-life based on degradation kinetics

• Proteasome inhibitor studies:

  • Treat samples with MG132 or other proteasome inhibitors

  • Compare ERF113 protein levels with and without inhibitor treatment

  • Similar to approaches used for studying EBF1/EBF2 stability in ethylene signaling

• Ubiquitination analysis:

  • Immunoprecipitate ERF113 using specific antibodies

  • Probe western blots with anti-ubiquitin antibodies

  • Identify ubiquitination sites through mass spectrometry

• Identification of regulatory E3 ligases:

  • Screen for E3 ligases that interact with ERF113 using co-immunoprecipitation

  • Confirm interactions using yeast two-hybrid or BiFC assays

  • Assess effects of candidate E3 ligases on ERF113 stability

• Environmental regulation of stability:

  • Compare protein turnover rates under different stress conditions

  • Correlate protein stability with transcriptional activity

  • Investigate how post-translational modifications affect protein half-life

These approaches can reveal mechanisms regulating ERF113 protein levels, which may be independent of transcriptional control.

What are the best practices for multiplexed detection of ERF113 with other proteins?

Simultaneous detection of ERF113 with other proteins provides valuable insights into co-localization and potential interactions:

• Multiplex immunofluorescence:

  • Select antibodies raised in different host species (e.g., rabbit anti-ERF113 with mouse anti-partner protein)

  • Use differentially labeled secondary antibodies (e.g., Alexa Fluor 488 and Alexa Fluor 594)

  • Include appropriate controls for each antibody separately

  • Use confocal microscopy for high-resolution co-localization analysis

• Sequential immunoprecipitation:

  • First immunoprecipitate with ERF113 antibody

  • Elute complexes and perform second immunoprecipitation with antibody against potential partner

  • Analyze resulting complexes to confirm direct or indirect interactions

• Proximity ligation assay (PLA):

  • Use primary antibodies from different species targeting ERF113 and potential interacting proteins

  • Apply species-specific PLA probes and perform ligations and amplifications

  • Quantify signals to assess proximity and potential interactions

• Mass spectrometry-based approaches:

  • Immunoprecipitate ERF113 complexes using validated antibodies

  • Identify interacting proteins through mass spectrometry

  • Confirm interactions using reciprocal immunoprecipitations

• Quantitative correlation analysis:

  • Quantify relative levels of multiple proteins across different conditions

  • Perform correlation analysis to identify co-regulated proteins

  • Establish potential functional relationships based on coordinated expression patterns

These approaches facilitate comprehensive analysis of ERF113 in its native protein interaction networks.

How should researchers interpret differences in ERF113 detection across experimental conditions?

Proper interpretation of ERF113 detection data requires careful consideration of several factors:

• Distinguishing technical from biological variation:

  • Include technical replicates to assess method reliability

  • Establish clear criteria for what constitutes significant changes in protein levels

  • Use appropriate statistical tests based on data distribution

• Normalizing protein quantification:

  • Select appropriate housekeeping proteins or total protein staining for normalization

  • Ensure normalization controls are not affected by experimental conditions

  • Consider using multiple normalization approaches to confirm findings

• Interpreting subcellular localization changes:

  • Quantify nuclear-to-cytoplasmic ratios rather than absolute intensities

  • Correlate localization changes with transcriptional activity measurements

  • Consider performing fractionation experiments to confirm imaging results

• Correlating with functional outcomes:

  • Connect changes in ERF113 levels/localization with downstream gene expression

  • Assess physiological responses correlated with protein changes

  • Similar to how GmERF113 levels correlate with drought resistance phenotypes

• Addressing conflicting results:

  • Consider post-translational modifications that might affect antibody recognition

  • Verify results using multiple antibodies targeting different epitopes

  • Integrate protein-level data with transcript measurements to identify discrepancies

What computational tools can assist in analyzing ERF113 binding sites from ChIP data?

Analysis of ERF113 binding sites requires specialized computational approaches:

• ChIP-seq analysis pipeline:

  • Use established tools like MACS2 for peak calling

  • Apply appropriate false discovery rate controls

  • Visualize binding profiles using genome browsers

• Motif discovery and analysis:

  • Search for enriched DNA motifs within binding regions using tools like MEME or HOMER

  • Compare identified motifs with known ERF binding sites (GCC-box and variants)

  • Analyze motif conservation across different experimental conditions

• Integration with genomic features:

  • Annotate binding sites relative to transcription start sites, gene bodies, and other genomic features

  • Correlate binding with epigenetic modifications using publicly available datasets

  • Identify co-occurring transcription factor binding sites

• Comparative analysis:

  • Compare ERF113 binding profiles with other ERF family members

  • Analyze binding site overlap between different stress conditions

  • Identify condition-specific and shared binding events

• Network construction:

  • Build gene regulatory networks based on binding data and expression profiles

  • Identify hub genes and regulatory modules

  • Predict indirect regulatory effects through network analysis

These computational approaches transform raw binding data into mechanistic insights about ERF113 function.